The role of nucleation in patterning microtubule networks

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Journal of Cell Science 111, 2077-2083 (1998) Printed in Great Britain © The Company of Biologists Limited 1998 JCS5004

COMMENTARY The role of nucleation in patterning microtubule networks A. Hyman and E. Karsenti Cell Biology Programme, EMBL, Meyerhofstrasse 1, Heidelberg 69117, Germany (e-mail: [email protected]; [email protected])

Published on WWW 15 July 1998

SUMMARY Control of microtubule nucleation is important for many microtubule dependent processes in cells. Traditionally, research has focused on nucleation of microtubules from centrosomes. However, it is clear that microtubules can nucleate from non-centrosome dependent sites. In this review

we discuss the consequences of non-centrosome dependent microtubule nucleation for formation of microtubule patterns, concentrating on the assembly of mitotic spindles.

INTRODUCTION

cell centrosomes. While text books generally show microtubules nucleated exclusively by centrosomes, in a variety of cells many microtubules are not anchored at the centrosome. For instance in migrating newt lung cells, 80-90% of the microtubules are not bound to the centrosome (Waterman-Storer and Salmon, 1997). In epithelial cells microtubules form bundles parallel to the apico-basal axis (Bacallao et al., 1989; Mogensen et al., 1989); During myogenesis the centrosomes are eliminated during formation of parallel bundles of microtubules in the developing myotubes (Tassin et al., 1985). In most of these cells it is not clear how non-centrosome microtubules arise. One possibility is that microtubules are centrosome nucleated, but that their connections with the centrosome are then severed, allowing the microtubules to be organized into different patterns. This phenomenon, initially observed in Xenopus egg extracts (Belmont et al., 1990), has now been seen in tissue culture (Keating et al., 1997). The discovery of katanin (McNally et al., 1996), a microtubule severing protein, suggests that it will soon be possible to investigate the role of severing in generating microtubule patterns. Centrosome-free microtubules can also arise from microtubule breakage (Waterman-Storer and Salmon, 1997). In this case microtubules are not severed at the centrosome but arise by breakage from preexisting microtubules. Perhaps the clearest example of microtubule severing is in the organization of microtubules in neurons. In these cells microtubules fill up the entire axon, while the centrosome is located in the cell body. How are the microtubules in the axons formed? Recent studies have shown that microtubules are first nucleated at centrosomes, released, and then carried down the axon by cytoplasmic dynein, perhaps anchored to the cell cortex (Ahmad et al., 1998; Ahmad and Bass, 1995). Thus in this instance centrosomes act as a microtubule generator (Ahmad et al., 1998), and motors then organize the microtubules into the correct distribution. Although non-centrosomal microtubules can be generated by

If one looks at the pattern of microtubules in a typical tissue culture cell, in general one will see microtubules distributed with their minus ends clustered together and their plus ends extending out into the cytoplasm. The canonical model for how such arrays are generated is that microtubules, nucleated at centrosomes, grow through the cytoplasm. Such growth patterns create radial arrays of microtubules extending from the centrosome. In other situations microtubules form non-radial patterns. For instance in mitotic spindles more microtubules extend from centrosomes towards chromosomes than away from chromosomes into the cytoplasm. The discovery of dynamic instability of microtubules in 1984 (Mitchison and Kirschner, 1984) suggested how centrosome-nucleated microtubules could form patterns (Kirschner and Mitchison, 1986). Dynamic instability describes the stochastic behaviour of microtubules in which individual microtubules transit between phases of growth and fast shrinkage. By such behaviour microtubules nucleating from centrosomes were proposed to act as searching devices (Holy and Leibler, 1994; Kirschner and Mitchison, 1986). Upon contact with capture sites they would be stabilized, forming patterns. This idea of selective stabilization of microtubules nucleated from centrosomes was a major step forward in understanding the morphogenetic properties of microtubules. It has been particularly useful in understanding how microtubules are stabilized by kinetochores during spindle formation, or how microtubule arrays are positioned in interphase by the use of cortical sites. The exact nature of nucleating centers or centrosomes has been a matter of controversy. We prefer the definition as a pair of centrioles surrounded by nucleating material (Fuller et al., 1992; Stearns and Winey, 1997). There are other kinds of nucleating centers, for example the spindle pole bodies in yeast, which have structures that are very different from that of somatic

Key words: Microtubule, Nucleation, Mitotic spindle, Centrosome

2078 A. Hyman and E. Karsenti severing or breaking of centrosomal-nucleated microtubules, a number of recent papers have reminded us that microtubules can also nucleate independently of centrosomes in cytoplasm. Observation of free nucleation in Xenopus extracts (Gard and Kirschner, 1987), fragments of cells without nuclei (Karsenti et al., 1984b; Maniotis and Schliwa, 1991; McNiven and Porter, 1988; Rodionov and Borisy, 1997) and normal tissue culture cells (Vorobjev et al., 1997; Yvon and Wadsworth, 1997) show that nucleation independent of centrosomes is a common occurrence. Nucleation of microtubules independently of centrosomes raises three major questions: (1) what is the mechanism of microtubule nucleation in the absence of centrosomes? (2) how is the nucleation controlled so that microtubules are nucleated only where they are needed? (3) how are non-centrosomal microtubules organized into patterns? MECHANISMS OF MICROTUBULE NUCLEATION In order to understand why some microtubules are nucleated by centrosomes whereas others originate from apparently undefined cellular domains, it is necessary to understand the mechanisms of microtubule nucleation. Microtubules are in dynamic exchange with a pool of soluble tubulin subunits. The assembly characteristics of pure tubulin in vitro can be defined by three ‘minimum concentrations’: (1) a concentration

between 0-10 µM, that allows transient growth of microtubules. At this concentration, microtubules exhibit dynamic instability, transiting between phases of growth and shrinkage. No assembly is possible in the absence of centrosomes or other nucleation sites. (2) A concentration in the order of 10-20 µM above which microtubules will grow indefinitely from centrosomes. Nucleation in the absence of centrosomes will still not take place. (3) A minimum concentration for free nucleation in the range of 20-40 µM (Bré and Karsenti, 1990; Fygenson et al., 1995; Mitchison and Kirschner, 1984) (Fig. 1a). Above the concentration for centrosome-independent nucleation, microtubules start to appear through self nucleation (Fig. 1a), resulting in a mixture of centrosome and non-centrosome nucleated microtubules. Pure tubulin nucleates at fairly high concentrations because elongation of the microtubule wall requires the formation of nucleation seed of about 12-15 dimers. At low concentrations the time required to form this stable nucleus is infinite and no elongation occurs (Fygenson et al., 1995; Mitchison and Kirschner, 1984). Above the minimum concentration, stable complexes form at a constant rate and the number of microtubules in the solution increases linearly as a function of time (Fygenson et al., 1995) until the polymer mass starts to deplete the free tubulin pool below the concentration required for formation of stable seed (Fig. 1b,c). The mechanisms by which centrosomes nucleate

a) [tub]dy < 10 µM

20 µM < [tub]fn

10 µM < [tub]ig < 20 µM

b) [tub]dy < 10 µM

10 µM < [tub]ig < 20 µM

20 µM < [tub]fn

c) MT number

Fig. 1. Concentration dependence of free versus centrosome induced nucleation in pure tubulin solutions. (a) The aster is nucleated by a centrosome. The given concentrations are roughly accurate. The length of microtubules are arbitrary, but given for infinite time. Above 10 µM they should have infinite length in the presence of an infinite supply of tubulin subunits. dy, dynamic instability regime; ig, infinite growth regime; fn, free nucleation regime. (b) Concentration dependent probability of assembly of a stable 12-15 mere nucleus supporting microtubule elongation in the absence of nucleating centers. (c) Microtubules form at a constant rate above the tubulin concentration at which stable 12-15 meres can form ([tub] >20 µM).

Time: 1 minute

2 minutes

3 minutes

Time

Microtubule nucleation 2079 microtubules at low tubulin concentration have recently been reviewed (Stearns and Winey, 1997). Much attention has focused on the role of γ-tubulin, a ubiquitous member of the tubulin family required for centrosome dependent nucleation in all species studied (Stearns and Winey, 1997). It is thought to bind to centrosomes and form a stable nucleation template for the addition of subsequent α-β dimers of tubulin, eliminating the requirement for the formation of the stable seed of 12-15 α-β dimers which occurs only above about 20 µM tubulin (Fig. 1). The tubulin concentration in cells is generally below that required for free nucleation of microtubules in vitro, suggesting that non-centrosomal nucleation must be driven by cellular factors. The mechanisms by which non-centrosomal microtubules nucleate fall into two classes: (1) nucleation sites not located at centrosomes (see Fig. 2). In this mechanism, dispersed nucleation sites provide a template to overcome the need for formation of a nucleation seed as with centrosomes. The obvious candidate for this molecule is γ-tubulin, implicated in the nucleation process from centrosomes, and many cells have a large cytoplasmic pool of γ tubulin. It is not known whether the cytoplasmic pool is active, but that it could drive dispersed nucleation is suggested by the fact that overexpression of γ tubulin in tissue culture cells drives the formation of ectopic microtubule nucleation. (2) Microtubule stabilizing proteins could favour the formation of a nucleation seed. Microtubule associated proteins (MAPs) are traditionally known as proteins which stabilize microtubules during growth (Mandelkow and Mandelkow, 1995). However, MAPs like tau or MAP2 also reduce the tubulin concentration required for nucleation in pure tubulin down to that required for nucleation by centrosomes (Bré and Karsenti, 1990) (Fig. 3). To date, we do not know whether non-centrosomal microtubules observed in vivo originate from spontaneous assembly due to the activity of MAPs or to nucleation by dispersed nucleation sites. CONTROL OF MICROTUBULE NUCLEATION When microtubules nucleate in the absence of centrosomes, the cell needs a way to control where and when this takes

b)

a)

Centrosome

-tubulin complex

place. There are a number of observations suggesting that nucleation can be controlled. For instance the assembly of microtubules in cytoplasts devoid of centrosomes increased dramatically when cells reached confluence, suggesting that the level of free nucleation could be regulated by cell-cell interactions (Karsenti et al., 1984b). Some insights into the regulation of non-centrosome nucleation have come from the study of spindle assembly in the absence of centrosomes, which occurs during formation of female meiotic spindle in animal cells and all spindles in plants (Endow and Komma, 1997; Gard, 1992; Lambert, 1993; Matthies et al., 1996). The steps with which a spindle forms without centrosomes in Xenopus best illustrate how non-centrosomal nucleation must be controlled in time and space for successful spindle assembly (Heald et al., 1997). (1) Control in time. During interphase, microtubules are nucleated both from centrosome and non-centrosome sites. In mitosis, centrosome-dependent nucleation continues, but non-centrosome nucleation is shut off, showing that non-centrosome nucleation is under control of the cell cycle (Verde et al., 1990). Cell cycle control of noncentrosome nucleation is seen in a number of embryonic systems for instance Caenorhabditis elegans embryos (Albertson, 1984; Hyman and White, 1987). (2) Control in space. Although a mitotic extract has no non-centrosome nucleation, non-centrosomal nucleation can be triggered by the addition of chromatin. However, non-centrosomal nucleation is seen only in the vicinity of the chromatin, nowhere else in the cytoplasm (Heald et al., 1997; Karsenti et al., 1984a). The mechanisms by which chromatin could stimulate nucleation remain obscure. One possibility is that the nucleation activity of dispersed nucleation complexes, such as the γ-tubulin complex, is regulated. Thus, non-centrosome nucleation templates would be active in interphase, turned off in mitosis, but activated locally in the vicinity of chromatin. Another possibility is that formation of a nucleation seed is favoured around chromatin, perhaps by the activation of MAPs (see Fig. 3). While nucleation may be controlled, another possibility is that nucleation is active throughout the cell cycle but in mitosis the nucleated microtubules are too unstable to grow. In this model, nucleation is uncontrolled, but microtubules would be stable in interphase, unstable in mitosis and stabilized locally in the

c)

Minus end directed motor

Fig. 2. Nucleation by γ-tubulin templates. Nucleation focused by the association of γ-tubulin templates with centrioles (a), nucleation by randomly distributed γ-tubulin templates (b), and self organization of randomly nucleated microtubules by minus end directed motors (c).

2080 A. Hyman and E. Karsenti Fig. 3. The effect of MAPs and microtubule destabilizing factors on centrosome dependent and free microtubule nucleation. In a tubulin solution that does not allow free nucleation, but centrosome dependent nucleation (left), adding MAPs like tau or MAP2 leads the appearance of free microtubules in addition to centrosome-nucleated ones (center). It is expected that in some cells where there are MAPs like tau and MAP2, it is the presence of destabilizing factors that prevent the occurrence of free microtubules (right).

[Tubulin] < [Tubulin]free nucleation

[Tubulin] < [Tubulin]free nucleation + MAPs

[Tubulin] < [Tubulin]free nucleation + MAPs + destabilizing factors

- Centrosomes

+ Centrosomes

vicinity of chromatin. We suspect that microtubule stability in vivo comes from the competing activities of microtubule destabilizing factors such as OP18/stathmin (Belmont and Mitchison, 1996; Marklund et al., 1996; Tournebize et al., 1997) and XKCM1 (a microtubule-based motor) (Walczak et al., 1996), and the stabilizing activities of MAPs (Fig. 3). If stabilizing factors were activated and destabilizing factors inactivated around chromatin, this would favour microtubule growth (Hyman and Karsenti, 1996). Support for such a hypothesis has come from analysis of the activity of one destabilizing molecule, OP18/stathmin, which is inactivated by chromatin (Andersen et al., 1997; Marklund et al., 1996). Depletion of this destabilizing factor from Xenopus cytoplasmic extracts leads to enhanced microtubule nucleation in the vicinity of chromatin (Andersen et al., 1997). Further experiments on the role of MAPs and nucleating factors in spindle assembly will be required to differentiate between these mechanisms.

ORGANIZATION OF RANDOMLY NUCLEATED MICROTUBULES While nucleation from centrosomes provides a built-in mechanism for focusing microtubules, the dispersed nucleation of microtubules leaves a problem of microtubule organization. For instance it is easy to understand how a bipolar spindle is formed between two centrosomes. Before mitosis the centrosome duplicates and each of the two centrosomes forms one of the spindle poles. Microtubules nucleated from centrosomes are captured by chromosomes and stabilized, forming a mitotic spindle. However, if microtubule nucleation is dispersed, such as in plant spindles or meiotic animal spindles, the situation is complex. It seems that in these cases, microtubules are organized in space through the activity of motors. It has been known for a long time that the stabilization of microtubules by heavy water or taxol could lead to the

Fig. 4. The kinetic dominance of - centrosome centrosome dependent versus free - Taxol nucleation during spindle assembly in (observed) Xenopus egg extracts. In the absence of centrosomes, chromosomes stimulate free nucleation around them. These microtubules are then sorted by motors into a bipolar spindle (top). When one + 1 centrosome centrosome is present, it nucleates - Taxol microtubules way before the appearance (observed) of free microtubules, creating an array of microtubules with homogeneous polarity, plus ends towards the chromosomes. The free microtubules that appear later are automatically oriented and dragged towards the centrosome along the + 1 centrosome centrosomal microtubules by dynein. The + Taxol more microtubules in this pole, the (predicted) strongest the effect. This prevents the appearance of a second pole (middle). Both previous situations have been 30 minutes 15 minutes 5 minutes observed. This model predicts that if free microtubules appear in the same time and in similar numbers to the centrosomal ones, a bipolar spindle could still form in the presence of a single centrosome. This could be tested by assembling spindles in the presence of small amounts of taxol (bottom).

Microtubule nucleation 2081

Actin dependent Contraction

Microtubule reorganization

Centriole Actin microfilament

-

-

+

+

Microtubule

Dynein

Tight and adherens junctions

Fig. 5. Establishment of a polarized array of microtubules in epithelial cells. Upon disruption of junctional complexes in cultures epithelial cells, the centrioles move towards the nucleus from where a radial array of microtubules emanates (top). Following the establishment of junctions, the centrioles split apart in a microtubule and actin-dependent way (middle). We propose that dynein, anchored at the level of the junctions pulls onto the microtubules, thereby pulling the centrioles apart. Probably, an actin-dependent contraction follows and in the same time, the dynein moves towards microtubule minus ends, organizing the microtubules into apico-basal bundles and raising up the junctions. Although this is still entirely hypothetical, this is just an extension of the self organization principle uncovered in the mechanism of mitotic spindle assembly.

generation of microtubule asters in the absence of centrosomes (De Brabander et al., 1981, 1986; Karsenti et al., 1984a). In the absence of centrosomes, a set of randomly growing microtubules can become organized into an astral array by minus or plus-end directed motors (Nédélec et al., 1997; Urrutia et al., 1992; Verde et al., 1991) (Fig. 2c). This shows that an astral array of microtubules can be generated by two independent pathways: through nucleation from a fixed point, a centrosome, or through the reorganization of randomly nucleated microtubules into an aster. Thus, formation of spindles in the absence of centrosomes provides a nice example in which spindle assembly is triggered by the control of nucleation around chromatin, followed by their organization into spindles by motors. CENTROSOME VS NON-CENTROSOME SPINDLE POLE FORMATION While it is clear in some systems that spindles can form in the absence of centrosomes (reviewed by Waters and Salmon, 1997;

Merdes and Cleveland 1997), in other systems centrosomes seem to be required. For instance, in grasshopper spermatocytes, micromanipulated chromosomes require centrosomes to form a spindle. Inhibition of centrosome duplication in sea urchin eggs (resulting in the presence of only one centrosome per egg) results in the formation of a monopolar spindle (e.g. Mazia, 1984; Sluder and Rieder, 1985). These experiments suggested that centrosomes direct spindle bipolarity. Recent experiments in Xenopus egg extracts suggest that the role of centrosomes may be more complex. As stated above, in Xenopus extracts spindles can form in the absence of centrosomes by microtubule nucleation around chromatin (Heald et al., 1996). Spindles will also assemble in Xenopus extracts in the presence of centrosomes (Lohka and Maller, 1985). Here, preventing centrosome separation will force these microtubules to become organized into a monopolar array (Sawin et al., 1992). However, removing the centrosome allows the formation of bipolar arrays (Heald et al., 1997). Thus in Xenopus, centrosomes are not required for bipolarity, but when present they are dominant and their number will control the number of spindle poles (Heald et al., 1997). Therefore, the original sea urchin experiments did not show that two centrosomes are required to make a bipolar spindle. Rather they showed that when centrosomes are present their number can control the number of spindle poles. How can a single centrosome prevent the establishment of a bipolar spindle in Xenopus extracts? We imagine two different ways this could work. (1) Differences in nucleation rates. Noncentrosome nucleation is very slow to initiate in egg extracts (several minutes) whereas centrosome dependent nucleation is almost immediate (Fig. 4). Why would the rate of nucleation from centrosomes be higher than that of free nucleation? This could be because the mechanisms of nucleation are different, for example, non-centrosome nucleation is non templated but relies on the formation of a nucleation seed (Fig. 1). (2) Local density of nucleating events. In this case, non-centrosome nucleation followed by elongation happens at random around chromatin. Therefore, the probability that enough microtubules are cross-linked by motors to generate a pole and become further stabilized together is low, leading to a time delay in the appearance of a visible pole (Fig. 4). By contrast, since the centrosome has many nucleating sites, it will nucleate many microtubules in a concentrated spot even if each nucleation event is infrequent. In both cases, the centrosome breaks the symmetry of microtubule distribution and all microtubules nucleated from non-centrosome sites around the chromosomes would then become oriented by motors relative to these preexisting microtubules and moved towards the centrosome (Fig. 4). We would predict that the dominance of centrosomes could be abolished by reducing the asymmetry of microtubule growth around chromosomes. For example small amounts of agents which trigger nucleation, such as taxol, would favour the rate of free nucleation, perhaps triggering bipolar spindle formation in the presence of a centrosome (Fig. 4). In fact in some situations, preventing centrosome separation does not prevent formation of two poles (Wilson et al., 1997). This could be because the density of microtubules or the rate of nucleation is higher in Drosophila embryos than in Xenopus extracts. In conclusion one of the interesting facts to emerge from the study of spindle assembly in meiosis is that whereas centrosomes are dispensable, when present they provide dominant sites for organization of microtubules. Thus they provide an added layer

2082 A. Hyman and E. Karsenti of regulation, allowing the determination of the position of the spindle poles. This is essential in systems in which spindle positioning is part of the differentiation process that takes place during cell division. Thus the centrosome can be positioned by the microtubules it nucleates that act as sensors of spatial cues, and then direct the organization of other microtubules in the cell from its location (Hyman and White, 1987). Most of the results obtained on the role of centrosome vs non centrosome components in microtubule nucleation were obtained in meiotic cells, and it is not clear to what extent the principles uncovered in meiotic systems may also apply also during spindle assembly in somatic cells. In at least one case bipolar spindles can assemble in the absence of centrosomes in tissue culture cells (Debec et al., 1982). However, as mentioned above, grasshopper spermatocytes require a centrosome to make a mitotic spindle. It seems possible that the requirement for centrosomes reflects the ability of noncentrosome microtubules to nucleate in cytoplasm. When noncentrosome microtubules can nucleate, centrosomes are not required, although they influence the position of the spindle when present. When non-centrosome microtubules cannot be formed by nucleation, centrosomes are required as a generator of microtubule polymer for spindle assembly. PERSPECTIVES In this review we have concentrated mainly on the role of nucleation in building a mitotic spindle during meiosis. We are beginning to understand some of the basic mechanisms by which microtubule distribution is controlled spatially and temporally during mitotic spindle assembly. As discussed in the Introduction, there are many complex microtubule arrangements in differentiated cells which are clearly noncentrosomal patterns, and at present we have little idea how these arrangements are controlled. The challenge is to understand how the principles elucidated from spindle assembly apply to these complex differentiated cell types. One of the important aspects will be to distinguish between centrosomes as microtubule generators, as in neurons, and noncentrosome nucleation in the cytoplasm. During meiotic spindle assembly, chromatin locally controls microtubule nucleation. In interphase, it is not clear what would control microtubule nucleation, but one possibility is the localization of enzymes which control the dynamics of microtubules to cellular sites as has been suggested for meiotic spindle assembly. Interestingly two cases have been documented suggesting different cellular domains have different properties. In neurons, a gradient of phosphorylation of tau extends down the neuron. This seems to be maintained actively by a phosphatase-kinase balance (Mandel and Banker, 1996). In one tissue culture line, plus end dynamics seem to be regulated differently depending on the cellular context (Waterman-Storer and Salmon, 1997). Obviously a lot more work needs to be done in this direction. Organization of non-centrosomal microtubules into patterns using motors provides a potentially powerful mechanism for making microtubule patterns. There are many different kinesins in differentiated cell types, which could organize microtubules into different patterns, depending on their properties. A good example is the rearrangement of microtubules in epithelial cells (Fig. 5). Epithelial cells are linked together by

junctions. When the junctions are broken, the cells loose their polarity. Under such conditions, microtubules become organized in a radial array originating from an ill defined point localized close to the nucleus (Fig. 5, top) (Bré et al., 1990). If one allows the cells to re-establish junctions and polarize again, microtubules reorganize so that they have a uniform orientation with their plus end facing the basolateral domain with some randomly oriented microtubules in the apical domain (Fig. 5, bottom) (Bacallao et al., 1989; Mays et al., 1994). How could microtubules become oriented in such a way? We envision two mechanisms by which they become oriented in epithelial cells. (1) The nucleating centers could be moved towards the apical domain of the cells, thereby orienting microtubule growth. This mechanism uses localization of nucleation sites to pattern the microtubule network. Actin may play an important role in this process (Buendia et al., 1990). γ-Tubulin has been localized where it is expected to be if involved in microtubule nucleation in epithelial cells (Meads and Schroer, 1995; Mogensen et al., 1997). However, there is no demonstration that the organization and orientation of microtubules is really defined by a localization of nucleating material yet. (2) Minus end directed motors like dynein could be positioned on the junctions of the cells. In this way, microtubules would be pulled away from the centrosome and moved, plus ends facing the basolateral domain of the cells (Karsenti et al., 1996). In summary, centrosomes provide an organization principle different from free nucleation by two virtues: (a) one centrosome can nucleate many microtubules originating from one point, whereas free nucleation has no organizing power in itself. (b) Centrosomes can nucleate under conditions where free nucleation is suppressed, providing dominant sites for microtubule organization. Nucleation in the absence of centrosomes requires organization principles such as motor interactions, but provides a potentially versatile system for making such patterns. Given the vast array of different microtubule patterns in diverse cell types, it is going to be important to understand the contribution of different forms of nucleation in generating microtubule patterns. REFERENCES Ahmad, F. G. and Bass, P. W. (1995). Microtubules released from the neuronal centrosome are transported into the axon. J. Cell Sci. 108, 27612769. Ahmad, F. G., Echeverri, J., Vallee, R. B. and Bass, P. W. (1998). Cytoplasmic dynein and dynactin are required for the transport of microtubules into the axon. J. Cell Biol. 140, 391-401. Albertson, D. G. (1984). Formation of the first cleavage spindle in nematode embryos. Dev. Biol. 101, 61-72. Andersen, S., Ashford, A. J., Tournebize, R., Gavet, O., Sobel, A., Hyman, A. and Karsenti, E. (1997). Mitotic chromatin regulates phosphorylation of Stathmin/OP18. Nature 389, 640-643. Bacallao, R., Antony, C., Dotti, C., Karsenti, E., Stelzer, E. H. K. and Simons, K. (1989). The subcellular organization of MDCK cells during the formation of a polarized epithelium. J. Cell Biol. 109, 2817-2832. Belmont, L. D., Hyman, A. A., Sawin, K. E. and Mitchison, T. J. (1990). Real-time visualization of cell cycle-dependent changes in microtubule dynamics in cytoplasmic extracts. Cell 62, 579-589. Belmont, L. and Mitchison, T. J. (1996). Identification of a protein that interacts with tubulin dimers and increases the catastrophe rate of microtubules. Cell 84, 623-631. Bré, M. H. and Karsenti, E. (1990). Effects of brain microtubule-associated

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